This invention relates to the use and production of immunoglobulins which activate trk neurotrophin receptors.
The neurotrophins are a family of small, homodimeric proteins which promote effects on distinct but partially overlapping sets of neurons (Levi-Montalcini, R., Science 237:1154-62 (1987); Barde, Y. A., Neuron. 2:1525-1534 (1989); Korsching, S., J. Neurosci. 13:2739-2748 (1993); Eide, F. F., Lowenstein, D. H. and Reichardt, L. F., Exp. Neurol. 121:200-214 (1993). For instance, nerve growth factor (NGF), a well characterized member of the neurotrophin family, functions as a target-derived molecule which aids in determining the level of innervation during development by regulating the survival and differentiation, including process outgrowth, of the innervating neuronal population. NGF has effects on apoptosis (programmed cell death) and influences many facets of neuronal development; for example, regulation of axon branching and of gene expression (Barde, 1989).
In addition to NGF, some other members of the neurotrophin family include brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). These various growth factors show selectivity with some overlap as to their responsive neurons in the peripheral nervous system (PNS) and central nervous system (CNS). For instance, neurons responsive to NGF include sympathetic neurons and neural crest sensory neurons in the PNS and basal forebrain cholinergic neurons, striatal cholinergic neurons and cerebellar Purkinje cells in the CNS. PNS neurons responsive to BDNF include placode-derived sensory neurons, neural crest sensory neurons, nodose ganglion neurons and spinal motoneurons. BDNF responsive neurons in the CNS include basal forebrain cholinergic neurons, proprioceptive trigeminal neurons, substantia nigra dopaminergic neurons, retinal ganglion neurons and facial motoneurons. NT-3 responsive neurons include sympathetic and sensory neurons in the PNS and basal forebrain cholinergic neurons and locus coeruleus neurons in the CNS. NT-4/5 responsive neurons in the PNS include sympathetic neurons, dorsal root ganglion neurons and nodose ganglion neurons. NT-4/5 CNS responsive neurons include basal forebrain cholinergic neurons and locus coeruleus neurons.
Redundancy of the neurotrophins and their receptors exists. For instance, trkA receptors bind the neurotrophins NGF, NT-3 and NT-4/5. The receptor trkb binds the neurotrophins BDNF, NT-3 and NT-4/5. The receptor trkC binds NT-3. Although neurotrophins often recognize more than one receptor, they may activate different receptors to different degrees. For example, NT-3 activates trkA moderately, and trkB and trkc strongly.
Neurotrophins are of interest as potential therapeutic agents for a variety of neurodegenerative and neurologic diseases. There are a number of disorders which may relate to suboptimal activity of trk receptors or which are associated with inappropriate activity or levels of neurotrophins. These neurologic disorders include Alzeimer""s Disease, Parkinson""s Disease, amyotrophic lateral sclerosis (ALS), cerebral ischemia, nerve tissue ischemia, peripheral neuropathy (especially toxic and diabetic peripheral neuropathies), nervous system cancer and epilepsy. Alzeimer""s Disease, Parkinson""s Disease, ALS, the ischemias, and the neuropathies can be broadly be classified as neurodegenerative disorders.
Parkinson""s Disease is also known as paralysis agitans and shaking palsy. It is a chronic, progressive central nervous system disorder characterized by muscular rigidity, tremors and slow purposeful movement as well as a decrease in purposeful movement. Classical Parkinson""s Disease is of unknown ideology. However, a Parkinson""s syndrome can be drug-induced or caused by poisonings, such as carbon monoxide poisoning, and also by infarction or death of cells in the vicinity of the basal ganglia. Such cell death can be caused by insufficient blood flow, tumors and head trauma. Additionally, a postencephalitic Parkinsonism is known. Parkinson""s Disease tends to have insidious onset and a slowly progressive course. It tends to become incapacitating after a number of years.
An additional group of neurogenic disorders includes ALS which is an example of a neurogenic muscular atrophy. Other examples are infantile and juvenile spinal muscular atrophy which, like ALS, are anterior horn cell degenerative diseases. ALS is also know as Lou Gherrig""s Disease and includes progressive spinal-muscular atrophy and progressive bulbar palsy. In ALS, muscular weakness and atrophy typically begin in the hands and spread to the forearms and the legs. Muscles fasiculations progressing to spasticity, increased tendon reflexes and extensor plantar reflexes are also characteristic. The afflicted individual generally dies within several years, typically surviving no more than five years. Variants of ALS include progressive spinal-muscular atrophy or Aran-Duchenne muscular atrophy and progressive bulbar palsy or Duchenne""s paralysis or labioglossolaryngeal paralysis.
Peripheral neuropathies and peripheral neuritis include a large group of sensory, motor, reflex and vaso-motor symptoms which may be single or in combination and which may be produced by disease of a single nerve (mononeuropathy), or two or more nerves in separate areas (mononeuritis multiplex), or many nerves simultaneously (polyneuropathy, polyneuritis, multiple peripheral neuritis or multiple peripheral neuropathy). The etiologies include collagen vascular conditions, infections, toxic agents, malignancies, metabolic and autoimmune causes. For example, a toxic peripheral neuropathy can be caused by the chemotherapeutic agent taxol which can disrupt axonal transport in peripheral neurons. This causes pain in the nerve distribution of the afflicted neurons. Diabetic neuropathy is another example of a peripheral neuropathy. In diabetic neuropathy, peripheral sensory and sympathetic neurons tend to be affected early in the course of the disease usually causing pain or decreased sensation.
Epilepsy or seizure disorder includes a variety of seizures characterized by recurrent paroxysmal cerebral dysfunction associated with sudden, usually brief, attacks of altered consciousness, motor activity, sensory phenomena or inappropriate behavior. Any recurrent seizure pattern can be called epilepsy. Convulsive seizures are probably the most common form of attack and they begin with loss of consciousness and loss of motor control followed by tonic or clonic jerking of the extremities. In about three quarters of seizure cases in adults, no clear etiology is identified and the seizure is termed xe2x80x9csymptomaticxe2x80x9d or xe2x80x9cidiopathic.xe2x80x9d Some of the known causes of seizures include cerebral trauma, tumors or other brain disease, CNS infections, hyperpyrexia such as that associated with acute infection or heat stroke, toxic agents, metabolic disturbances, cerebral infarction or hemorrhage, cerebral hypoxia and a variety of other causes. Drug therapy with anticonvulsive agents is frequently effective.
There has been some exploration of the possibility of using neurotrophins to treat disease. For example, in animal models of both taxol toxic and diabetic peripheral neuropathies, NGF was used as a therapy with varying degrees of success. See Eide, et al. at page 206. However, because of the redundancy of neurotrophin-receptor recognition and because of variability in the strength of activation of their corresponding receptors, neurotrophins present complex problems as potential therapeutic agents. Additionally, several physiologic mechanisms exist for rapidly clearly neurotrophins from the circulation, including the presence of receptor-like binding proteins whose main function may be to sequester the neurotrophins. Additionally neurotrophins are difficult to prepare in quantity because of complex requirements for post-translational processing and their lability to proteases.
The importance of the neurotrophins has stimulated interest in their receptors and signal transduction mechanisms. Most approaches have focused on the pheochromocytoma cell line PC12, which is transformed into a sympathetic neuron-like cell when exposed to NGF (Greene, L. A. and Tischler, A., Proc. Natl. Acad. Sci. USA 73:2424-2428 (1976)). NGF binding studies indicated that NGF could bind to at least two different sites, a low affinity or fast-dissociating binding site, and a high affinity or slow-dissociating binding site (Sutter, et al., J. Biol. Chem. 254:5972-5982 (1979); Schechter, A. L. and Bothwell, M. A., Cell., 24, 867-874 (1981)).
A receptor for NGF, known as the low affinity nerve growth factor receptor (LNGFR), has been cloned from rat and human sources (Radeke, et al., 325:593-7 (1987); Johnson, et al., Cell. 47:545-554 (1986)). It is a transmembrane glycoprotein of 75,000 daltons, and it is expressed in many neuronal and nonneuronal cell types (Wyatt, et al., Neuron. 4:421-7 (1990); Wheeler, E. F. and Bothwell, M., J. Neurosci. 12:930-45 (1992)). However, LNGFR was unable to bind NGF with a high affinity in transfected fibroblastic cell lines (Radeke, et al., 1987).
Crosslinking studies (Hosang, M. and Shooter, E. M., J. Biol. Chem. 260:655-662 (1985)), biochemical characterization (Meakin, S. O. and Shooter, E. M., Neuron. 6:153-163 (1991); Radeke, M. J. and Feinstein, S. C., Neuron. 7:141-50 (1991)), and binding and culture studies with the anti-LNGFR polyclonal antibody REX (Weskamp, G. and Reichardt, L. F., Neuron. 6:649-63 (1991)) implied that another receptor for NGF is expressed by PC12 cells. That receptor has since been identified as the receptor tyrosine kinase p140trk or trkA (Kaplan, et al., Science. 252:554-8 (1991); Klein, et al., Cell. 65:189-97 (1991)).
TrkA is expressed in sensory and sympathetic neurons in the peripheral nervous system, and basal forebrain neurons in the central nervous system, all cell types which show responses to NGF (Martin-Zanca, et al., Genes Dev. 4:683-94 (1990); Schecterson, L. C. and Bothwell, M., Neuron. 9:449-63 (1992); Holtzman, et al., Neuron. 9:465-78 (1992)). Biochemical studies have demonstrated that trkA is phosphorylated in response to NGF (Kaplan, et al., Nature 350:158-60 (1991); Klein, et al., (1991); Jing, et al., Neuron. 9:1067-79 (1992)), and subsequently trkA activates several signal transduction pathways (Vetter, et al., Proc. Natl. Acad. Sci. USA 88:5650-4 (1991); Loeb, et al., Neuron. 9:1053-65 (1992); Obermeier, et al., Embo. J. 12:933-941 (1993)).
TrkB and trkC, two receptors closely related to trkA, have been isolated and can respond to other members of the neurotrophin family (Martin-Zanca, et al., Mol. Cell. Biol. 9:24-33 (1989); Klein, R., et al., Embo. J. 8:3701-9 (1989); Middlemas, et al., Mol. Cell. Biol. 11:143-53 (1991); Lamballe, et al., (1991)).
The invention relates to the discovery that selected immunoglobulins can activate trk receptors and mimic the actions of neurotrophins. Included in the invention are methods for activating a trk receptor comprising exposing cells having the trk receptor to a multivalent immunoglobulin which binds to the receptor and activates the receptor. The trk receptor is any of a number of tyrosine kinase receptors, and preferably it is any of trkA, trkB, and trkC.
Activation of the receptor can be noted by a variety of means including increased phosphorylation of the receptor, increased phosphorylation of protein substrates that are phosphorylated in response to activation of the receptor, and promotion of the effector function or outcome of receptor activation. Examples of such functions or outcomes include promotion of neuronal survival and promotion of neuronal differentiation including neurite outgrowth.
The multivalent immunoglobulin is preferably bivalent, although it can be polyvalent. Typically the immunoglobulin is a monoclonal antibody.
The invention includes methods of therapy for neurologic disorders associated with suboptimal activity of a trk receptor. Such disorders include Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis (ALS), cerebral ischemia, nerve tissue ischemia, peripheral neuropathy, particularly toxic and diabetic peripheral neuropathy, nervous system cancer, and epilepsy. Examples of nervous system cancer are primitive neuroectodermal tumors, neuroblastomas, medulloblastomas, ganglioneuromas, Ewing""s sarcoma, gliomas, glioblastomas, and astrocytomas.
The therapeutic method includes administering to a mammal having the disorder a therapeutically effective amount of a multivalent immunoglobulin of the invention. Usually the mammal is a human, although veterinary use is also contemplated. The administration is preferably parenteral, especially intraventricular, intravenous or intramuscular. The effective amount is an amount sufficient to cause a desired effect such as a therapeutic benefit. Typically, the dosage is selected from the range of from about 1 xcexcg/kg to about 1 mg/kg body weight of the recipient mammal for a polyclonal antibody. The range for a monoclonal antibody is usually about 5% to about 10% of the polyclonal antibody range.
A method for diagnosing a neurologic disorder associated with suboptimal activity of a trk receptor is also supplied. The method includes obtaining a nerve cellular sample, exposing the sample to a multivalent immunoglobulin which (1) binds to the receptor and (2) activates the receptor, and assaying the sample for (1) binding to the immunoglobulin and (2) activation of the receptor.
Also provided is a method for determining whether cellular material has a trk receptor. This method includes exposing the cellular material to a multivalent immunoglobulin which both binds to and activates the receptor and assaying the cellular material for binding to the immunoglobulin and activation of the receptor.
Thus, the invention provides immunoglobulins that mimic the actions of neurotrophins. Appropriately designed immunoglobulins able to dimerize the neurotrophin-receptors can activate these receptors and serve as neuronal survival and differentiation-promoting agents. Such immunoglobulins are easier to prepare, are more stable, and are likely to be longer acting than neurotrophins. In some cases immunoglobulins can be designed to act with greater selectivity than certain neurotrophins, several of which recognize more than one receptor. Bifunctional organic molecules that bind neurotrophin receptors are likely to also activate these receptors.
Immunoglobulins able to bind and cross-link the trk family of neurotrophin-receptors are shown herein to activate these receptors and produce consequences in neurons indistinguishable from exposure to a neurotrophin. Bivalent or polyvalent antibodies or immunoglobulins are required. Observed activation sequelae include tyrosine phosphorylation of the trk-receptor, tyrosine phosphorylation of the protein substrates that are phosphorylated as a response to the neurotrophin, promotion of neuronal survival, and promotion of neuronal differentiation including neurite outgrowth. Thus, the immunoglobulins mimic the actions of the neurotrophins.
Also provided are immunoglobulins, and methods of use, which are monovalent and which bind to and prevent activation of trk receptors. Such immunoglobulins and methods block the relevant receptor and prevent the receptor""s effect or function from occurring.
Immunoglobulins can be designed to recognize only single receptors. Included herein are data supporting detection of differentially spliced exons encoding extracellular domains in trk. Immunoglobulins recognizing specific receptor isoforms can be prepared. Immunoglobulins have different, and in some cases more restricted, specificities that are useful for targeting the therapy.
Immunoglobulins, especially monoclonal antibodies, are easy to prepare and purify. They can be designed to eliminate problems of antigenic responses or complement activation. They have considerable therapeutic potential in general. Additionally, they may be more efficiently delivered to neurons than are neurotrophins. Immunoglobulins would not be cleared as rapidly by their respective receptors and they are comparatively easy to prepare and purify. Also they are resistant to most proteases.